Iron is an essential element for nearly all forms of life. One of the challenges for organisms is the acquisition of iron due to its propensity to oxidize in aerobic environments and the extreme insolubility of ferric iron. As a result, organisms have evolved elaborate mechanisms for acquiring and storing iron. These mechanisms must be tightly regulated, however, due to the toxicity of free iron through its ability to catalyze the generation of free radicals through the Fenton reaction. In fact, aberrant iron regulation is associated with a variety of diseases and disorders in humans, including hemochromatosis, sideroblastic anemias, and Friedrich's ataxia, to name a few. Animals regulate iron uptake and storage primarily through the action of iron regulatory proteins (IRP), a family of sequence- specific, RNA binding proteins. IRPs regulate the synthesis of ferritin and transferrin receptor, proteins that serve in iron storage and iron transport, respectively. Through this regulation, animal cells are able to maintain iron homeostasis. IRPs also regulate the synthesis of proteins that are involved in heme biosynthesis and energy production. Thus the role of IRPs in cellular physiology is broader than simply iron regulation. There are two IRP family members, called IRP1 and IRP2. IRP1 is a bifunctional protein having the aforementioned activity as a RNA binding, gene regulator, or as the cytosolic isoform of aconitase. These activities are mutually exclusive and require the assembly and disassembly of a [4Fe-4S] cluster in the protein. Therefore, the activity of IRP1 and the regulation of iron in animals is dependent on the reversible assembly of an Fe- S cluster in this protein. In the proposed studies, we will define the mechanism and factors involved in the assembly/disassembly of the Fe-S cluster in IRP1. We will use a combination of molecular genetic, genetic and biochemical techniques in the yeast, Saccharomyces cerevisiae, to accomplish our goals. Our specific aims are to: 1) Define the mechanism of Fe-S cluster assembly in IRP1; 2) Determine the mechanism by which iron disrupts IRE/IRP1 complexes; 3) Investigate the process of Fe-S cluster disassembly in IRP1. The completion of these studies will help us to understand how organisms utilize Fe-S clusters as sensors of cellular iron status and oxidant levels as well as giving us insight into the fundamental question of Fe-S cluster assembly.